Studies On The Development Characteristic And Forming Mechanism Of Stolon In Tulipa Edulis(Miq.)Baker

Tulipa edulis(Miq.)Baker is an important medicinal plant of the genus Tulipa from the Liliaceae.Guangcigu,dried bulb of T.edulis without tomentum,possesses the efficacy of detoxicating and resolving a mass,activating blood circulation and dissipating blood stasis,and has been widely used for the treatment of dysentery,gout,sore throat as well as animal bites.At present,it is also an important medicine for the treatment of a variety of tumors such as throat cancer,lymphoma,and breast cancer.Stolon was an important reproductive organ of T.edulis for its rapid propagation.In addition to as an important asexual reproductive organ,stolon is also a vital mark of evolution and ecological adaption.So it is extraordinarily meaningful to explore the development characteristic and forming mechanism for clarifying its position in evolution,ecological adaptation,and for efficient growth regulation.In order to clarify the forming mechanism,experments were conducted to optimize best induction conditions of stolon.Based on this,structctural botany,plant physiology,molecular biology and bioinformatics are synthetically applied to explore the development characteristic and forming mechanism in current project.The morphological characteristic of stolon formation will be observed,and the physiological and biochemical changes will be measured in the dynamic process of stolon formation and development.Meanwhile,Transcriptome Sequencing and microRNA analysis were studied during stolon foramtion.This project will provide the scientific basis for the mechanism of stolon formation and stolon development.,and for further growth regulation,and for research of similar structure in other plants of Liliaceae.The main contents and results of this study are as follows:(1)The present study was conducted to explore the effects of differrent grades of planted bulb,cultured densities,cultured media,cultured depths and water treatments on T.edulis growth and stolon emergence condition.The result showed that the 2nd,the 3rd and the 4th grade bulbs are more applicable as materials for inducing stolon emergence,and the 3rd and the 4th grade bulbs are suitable to be reproductive materials for bulbs.T.edulis cultured with high density facilitated improving yield and inducing stolon emergence.Y+T treatment saved costs and was benefit for stolon induction and for maintaining T.edulis yield.T.edulis under the treatments of cultured depth 0.5 cm and 5 cm were regenerated in stolon-replacement manner,which was suitable for stolon induction,and meanwhile the treatment of cultured depth 5 cm was adopted to use for increasing production.Control and AWD were conducive to stolon indution,and AWD regime can be an effective means to promote water productivity and bulb production.(2)Based on stolon induction,the morphological characteristics of stolon formation were observed under the microscope through morphological anatomy and paraffin section.We found that the stolon was formed in the stem base near the side of bulb basal plate,and that the stolon bulged out from the bottom of stem base and extended to a larger extent.It shows roughly cylindrical and smooth in appearance,and has no visible node,intemode or adventitious root.Meristematic cells in the location where stolon of T.edulis occurred divide anticlinally and periclinally to develop a raised growth cone.Meanwhile,bud primordium growed with cell division to initiate stolon emergence.In the later period of stolon formation bud primordium develops a bud,and growth cone differentiates multiple primordiums,which led to stolon elongation and hence prepared for new bulb formation.Procambial strand is flourishing during stolon formation and it benefits for the transportation of water and nutrient.(3)The dynamic changes in carbohydrates and related enzymes,proteins and endogenous hormones during stolon formation in T.edulis were investigated.The results showed that the content of total soluble sugar,sucrose,reducing sugar,fructose,and starch were all significantly enhanced in the middle period when stolon emerged and maintained at relatively high levels until the later period of stolon formation,while protein content decreased during stolon formation.The activities of amylase(AMY),sucrose phosphate synthase(SPS),and sucrose synthase(SS)peaked during the initial period and were negatively correlated with soluble sugars.However,adenosine diphosphoglucose pyrophosphorylase(AGPase)activity increased as stolon formation progressed,and the changes in soluble starch synthase(SSS),granule-bound starch synthase(GBSS)activities presented a single peak,reaching their maximums in the middle period of stolon formation.AGPase,SSS and GBSS activities were all positively related to starch content.The gibberellin(GA)and zeatin riboside(ZR)contents attained their maximums in the initial period and then declined until the later period of stolon formation.Indole-3-acetic acid(IAA)and abscisic acid(ABA)remained at high levels during the initial and middle period and decreased significantly during the later period of stolon formation,inversely to the ratio of ABA/IAA.In conclusion,the accumulation of soluble sugars and starch via various enzymes,together with high level of IAA and a low ABA/IAA ratio,play an important role in stolon emergence,and increasing sucrose and reducing sugar content may also represent signal messengers that trigger stolon emergence.(4)The dynamic changes in carbohydrates and related enzymes,protein and endogenous hormones during T.edulis stolon development were investigated.The results showed that soluble sugar levels were basically declining,whereas starch and protein content rose continuously during stolon development.The adenosine diphosphoglucose pyrophosphorylase(AGPase)activity peaked at the initial swelling stage and stayed a relative high level at the middle swelling stage;sucrose synthase(SS),soluble starch synthase(SSS),and granule-bound starch synthase(GBSS)activities followed the same law that showed rising trends during stolon development.SS activity was significantly inversely related to sucrose content but had significantly positive relations with starch content,SSS,GBSS activities.Gibberellin(GA),indole-3-acetic acid(IAA),and zeatin riboside(ZR)peaked at the initial swelling stage and maintained high levels at the middle swelling stage;they then decreased significantly at the later swelling stage.A substantial increase was observed in abscisic acid(ABA)content until the middle swelling period,followed by a significant reduction in the later swelling period of stolon development.The ratios of ABA to IAA,GA,and ZR reached their lowest levels at the initial swelling stage.In conclusion,T.edulis stolon development is a process of new bulb morphogenesis along with the starch accumulation catalyzed by AGPase,SSS,and GBSS,using the product of sucrose cleavage caused by SS.Low ABA content facilitated the initial swelling of stolon together with the minimum ratios of ABA to IAA,GA,and ZR.The GA,IAA,and ZR of high-content,soluble sugars worked more efficiently to induce new bulb formation.(5)Transcriptome sequencing analysis of T.edulis stolon formation was performed via RNA-seq sequencing technology.15.49 Gb raw data was obtained during sequencing and 74,006 Unigne was acquired through splicing.From these sequences 2,484 SSR markers were screened.5,119 genes showed differential expression in at least a group of three developmental stages of stolon by contrasting the gene expression abundance of three transcriptome libraries.With GO,COG,KEGG database for differential gene annotation,functional annotation showed that these differentially expressed genes mainly related to material and energy metabolism,hormone signaling,cell growth,transcriptional regulation and many other physiological and biochemical processes.In addition,validation of quantitative real time PCR in differential gene expression was consistent with transcriptome data,which further testified the differentially expressed genes involved in the stolon formation.(6)High-throughput Illumina sequencing technology was used in the sequencing of stolon formation.12,890,912,12,182,122,12,061,434 clean reads were acquired in prophase,metaphase,and anaphase of stolon formation,respectively.In the reads of all miRNA,88 conserved miRNAs were identified,and 70 unique sequences were predicted to be the new miRNAs.The target genes of these miRNAs were predict,and 122 miRNAs predicted 531 target genes.Function prediction and annotation of target genes with GO,COG and KEGG databases contributed to a better exploration of gene function.miRNAs and their target gene expression profiles were further analyzed at different developmental stages during stolon formation.The results showed that miR165,miR396,miR1 886.2,miR2094,miR397 may play an important role in the regulation of stolon formation.(7)Expression levels of related genes encoding enzymes involved in sugar synthesis and carbohydrate metabolism verified dynamic changes of related enzymes activities in sugar synthesis and carbohydrate metabolism via the transcriptome and RT-PCR technology analysis.The genes regulating cell growth improved their expression level during stolon formation for participation in cell division and growth.Moreover,significant difference were observed in the expression level of genes involved in auxin polar transport,which participated in stolon formation.ath-miR165a,zma-miR396g-5p and ath-miR1 886.2 may be important regulatory factors during stolon formation.The target genes of zma-miR396g-5p、osa-miR2094-5p、ptc-miR7839 involved in carbohydrate transport and metabolism.